Massimo Tortarolo

Transgenic SOD1 G93A mice develop reduced GLT-1 in spinal cord without alterations in cerebrospinal fluid glutamate levels.

Glutamate‐induced excitotoxicity is suggested to play a central role in the development of amyotrophic lateral sclerosis (ALS), although it is still unclear whether it represents a primary cause in the cascade leading to motor neurone death. We used western blotting, immunocytochemistry and in situ hybridization to examine the expression of GLT‐1 in transgenic mice carrying a mutated (G93A) human copper–zinc superoxide dismutase (TgSOD1 G93A), which closely mimic the features of ALS. We observed a progressive decrease in the immunoreactivity of the glial glutamate transporter (GLT‐1) in the ventral, but not in the dorsal, horn of lumbar spinal cord. This effect was specifically found in 14‐ and 18‐week‐old mice that had motor function impairment, motor neurone loss and reactive astrocytosis. No changes in GLT‐1 were observed at 8 weeks of age, before the appearance of clinical symptoms. Decreases in GLT‐1 were accompanied by increased glial fibrillary acidic protein (GFAP) levels and no change in the levels of GLAST, another glial glutamate transporter. The glutamate concentration in the cerebrospinal fluid (CSF) of TgSOD1 G93A mice was not modified at any of the time points examined, compared with age‐matched controls. These findings indicate that the loss of GLT‐1 protein in ALS mice selectively occurs in the areas affected by neurodegeneration and reactive astrocytosis and it is not associated with increases of glutamate levels in CSF. The lack of changes in GLT‐1 at the presymptomatic stage suggests that glial glutamate transporter reduction is not a primary event leading to motor neurone loss.

Functional alterations of the ubiquitin-proteasome system in motor neurons of a mouse model of familial amyotrophic lateral sclerosis†

In familial and sporadic amyotrophic lateral sclerosis (ALS) and in rodent models of the disease, alterations in the ubiquitin-proteasome system (UPS) may be responsible for the accumulation of potentially harmful ubiquitinated proteins, leading to motor neuron death. In the spinal cord of transgenic mice expressing the familial ALS superoxide dismutase 1 (SOD1) gene mutation G93A (SOD1G93A), we found a decrease in constitutive proteasome subunits during disease progression, as assessed by real-time PCR and immunohistochemistry. In parallel, an increased immunoproteasome expression was observed, which correlated with a local inflammatory response due to glial activation. These findings support the existence of proteasome modifications in ALS vulnerable tissues. To functionally investigate the UPS in ALS motor neurons in vivo, we crossed SOD1G93A mice with transgenic mice that express a fluorescently tagged reporter substrate of the UPS. In double-transgenic Ub(G76V)-GFP /SOD1G93A mice an increase in Ub(G76V)-GFP reporter, indicative of UPS impairment, was detectable in a few spinal motor neurons and not in reactive astrocytes or microglia, at symptomatic stage but not before symptoms onset. The levels of reporter transcript were unaltered, suggesting that the accumulation of Ub(G76V)-GFP was due to deficient reporter degradation. In some motor neurons the increase of Ub(G76V)-GFP was accompanied by the accumulation of ubiquitin and phosphorylated neurofilaments, both markers of ALS pathology. These data suggest that UPS impairment occurs in motor neurons of mutant SOD1-linked ALS mice and may play a role in the disease progression.

Glutamate AMPA receptors change in motor neurons of SOD1G93A transgenic mice and their inhibition by a noncompetitive antagonist ameliorates the progression of amytrophic lateral sclerosis-like disease

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder involving the selective degeneration of motor neurons. In a small proportion of patients, ALS is caused by mutations in copper/zinc superoxide dismutase (SOD1), and mice overexpressing SOD1G93A mutant develop a syndrome that closely resembles the human disease. Excitotoxicity mediated by glutamate AMPA receptors has been suggested to be implicated in the selective susceptibility of motor neurons occurring in ALS. In SOD1G93A mice, we found that levels of GluR2 AMPA subunit, which plays a pivotal role in the maintenance of calcium impermeability of AMPA receptors, are decreased in spinal motor neurons before symptom onset in concomitance with a modest increase of GluR3 expression, a calcium‐permeable AMPA subunit. This effect can result in a higher number of calcium‐permeable AMPA receptors on motor neurons of SOD1G93A mice, predisposing these cells to be injured by AMPA‐mediated glutamate firing. In support of this, we showed that treatment with a new noncompetitive AMPA antagonist, ZK 187638, partially protected motor neurons, improved motor function, and prolonged the survival of SOD1G93A mice.

LACK OF APOPTOSIS IN MICE WITH ALS

be detected by this method. However, sublimon-type templates give equally prominent products. Our findings imply that the background of sublimon-derived products generated from control templates makes LX-PCR unreliable as a sole diagnostic method for detecting deleted mtDNAs, except in the case of deletions representing a substantial fraction of mtDNA molecules in a given DNA preparation. We would thus recommend routine serial dilution of all DNA samples to test for the meaningful presence of deleted mtDNA molecules when using LX-PCR, and ideally the verification of all positive findings by Southern blot analysis, before a diagnostic conclusion is reached. Published claims, based exclusively on LX-PCR analysis, that deleted mtDNAs accumulate to high levels in aging and in many disease states , need to be critically re-evaluated in the light of our findings. Acknowledgments We thank M. Niittylahti and O.Lumme for technical assistance, and P. Rustin, I. Holt, S. Khogali and N.-G. Larsson for discussions. This work was supported by grants from the Finnish Academy, Muscular Dystrophy Group, Royal Society, Tampere University Hospital Medical Research Fund, Yrjö Jahnsson Foundation, Finnish Foundation of Alcohol Research and the Pirkanmaa Region Fund of the Finnish Cultural Foundation.

Activation of the p38MAPK cascade is associated with upregulation of TNF alpha receptors in the spinal motor neurons of mouse models of familial ALS

Phosphorylated p38 mitogen-activated protein kinase (p38MAPK), but not activated c-jun-N-terminal kinase (JNK), increases in the motor neurons of transgenic mice overexpressing ALS-linked SOD1 mutants at different stages of the disease. This effect is associated with a selective increase of phosphorylated MKK3-6, MKK4 and ASK1 and a concomitant upregulation of the TNFalpha receptors (TNFR1 and TNFR2), but not IL1beta and Fas receptors. Activation of both p38 MAPK and JNK occurs in the activated microglial cells of SOD1 mutant mice at the advanced stage of the disease; however, this effect is not accompanied by the concomitant activation of the upstream kinases ASK1 and MKK3,4,6, while both the TNFRs are overexpressed in these cells. No changes of the upstream p38MAPK cascade kinases or TNFRs occur in reactive astrocytes. These findings highlight the activation of a selective intracellular signaling pathway in the motor neurons of SOD1 mutant mice, which is likely implicated in their death.

Mutant Copper-Zinc Superoxide Dismutase (SOD1) Induces Protein Secretion Pathway Alterations and Exosome Release in Astrocytes: IMPLICATIONS FOR DISEASE SPREADING AND MOTOR NEURON PATHOLOGY IN AMYOTROPHIC LATERAL SCLEROSIS*

Background: The mechanism by which astrocytes contribute to disease progression in mutant SOD1 mouse models of ALS is not known. Results: Mutant SOD1 astrocytes release mutant SOD1-containing exosomes that are toxic for motor neurons. Conclusion: Astrocyte-derived exosomes may have a role in disease spreading and motor neuron pathology. Significance: New therapeutic approaches should target exosomes to contain disease progression. Amyotrophic lateral sclerosis is the most common motor neuron disease and is still incurable. The mechanisms leading to the selective motor neuron vulnerability are still not known. The interplay between motor neurons and astrocytes is crucial in the outcome of the disease. We show that mutant copper-zinc superoxide dismutase (SOD1) overexpression in primary astrocyte cultures is associated with decreased levels of proteins involved in secretory pathways. This is linked to a general reduction of total secreted proteins, except for specific enrichment in a number of proteins in the media, such as mutant SOD1 and valosin-containing protein (VCP)/p97. Because there was also an increase in exosome release, we can deduce that astrocytes expressing mutant SOD1 activate unconventional secretory pathways, possibly as a protective mechanism. This may help limit the formation of intracellular aggregates and overcome mutant SOD1 toxicity. We also found that astrocyte-derived exosomes efficiently transfer mutant SOD1 to spinal neurons and induce selective motor neuron death. We conclude that the expression of mutant SOD1 has a substantial impact on astrocyte protein secretion pathways, contributing to motor neuron pathology and disease spread.

Differential Expression of S100β and Glial Fibrillary Acidic Protein in the Hippocampus after Kainic Acid-Induced Lesions and Mossy Fiber Sprouting in Adult Rat

The expression of S100beta and glial fibrillary acidic protein (GFAP) was analyzed following bilateral injection of kainic acid (KA), a glutamate derivative, into the CA3 region of the adult rat hippocampus. This treatment produces a progressive degeneration of the pyramidal neurons of the hippocampus while sparing the granule cells of the dentate gyrus which undergo sprouting of their axons in the supragranular layer. Messenger RNA and protein levels were measured, by Northern blot and ELISA, in the hippocampus of lesioned and sham-operated rats 1, 7, and 30 days after KA injection. A significant increase of GFAP and its mRNA was demonstrated at each time point, whereas S100beta mRNA levels were significantly enhanced only 30 days after the KA injection and the levels of S100beta protein remained unchanged at all time points. However, when analyzed by immunohistochemistry the S100beta showed clear changes in its expression and distribution depending on the region considered. One month after KA injection, S100beta immunoreactivity was considerably reduced in the stratum radiatum of CA3 region, but there was increased S100beta immunoreactivity in the stratum moleculare. In particular, a notable band of S100beta positive, hypertrophic astrocytes appeared in the supragranular layer of the dentate gyrus where the sprouting of mossy fiber collaterals was detected by Timms staining. These data show for the first time that an increase in S100beta expression in subpopulations of reactive astrocytes may be involved in the structural reorganization of the hippocampus following KA-induced neurodegeneration.

Expression of glutamate receptor subtypes in the spinal cord of control and mnd mice, a model of motor neuron disorder

We studied the expression and distribution of glutamate receptor subtypes in the spinal cord of mnd mice, a model of motor neuron disorders and neuronal ceroid lipofuscinosis, and control mice using immunocytochemistry and in situ hybridization. The constitutive subunit of the NMDA ionotropic glutamate receptor, NMDAR1, was expressed in all neurons of the grey matter and was not modified in the spinal cord of mnd mice in either its normal or phosphorylated form. The immunoreactivity of GluR2, but not its mRNA, was increased mainly in the substantia gelatinosa both in presymptomatic and in 8‐month‐old symptomatic mice, suggesting compensatory changes aimed at reducing the Ca2+ permeability of the receptor channel. In spinal cord of mnd mice, mRNA, and protein levels of GluR3 were low only at the symptomatic stage, possibly as a consequence of motor neuron dysfunction. This was not due to motoneuron degeneration, because the number of choline acetyltransferase (ChAT) immunopositive lumbar motor neurons and the ChAT activity in the spinal cord and hind leg muscles of symptomatic mnd mice were no different from control mice. GluR4 mRNA was increased throughout the grey matter, presumably in relation to the marked microglia activation reported in the grey matter of the lumbar spinal cord in mnd mice. These changes in ionotropic glutamate receptors may alter glutamatergic neurotransmission and play some role in the pathology of mnd mice.

Serotonin Inhibition of the NMDA Receptor/Nitric Oxide/Cyclic GMP Pathway in Rat Cerebellum: Involvement of 5-Hydroxytryptamine2C Receptors

Abstract: Previous studies have shown that 5‐hydroxytryptamine (5‐HT) can potently inhibit glutamatergic transmission in rat cerebellum through the activation of multiple 5‐HT receptors. The aim of this study was to subclassify the 5‐HT2 receptor mediating inhibition of the cyclic GMP response elicited by N‐methyl‐d‐aspartate in adult rat cerebellar slices. Seven receptor antagonists, endowed with relative selectivities for the 5‐HT2A, 5‐HT2B, and 5‐HT2C subtypes, differentially affected the inhibition by (±)‐1‐(2,5‐dimethoxy‐4‐iodophenyl)‐2‐aminopropane of the cyclic GMP response, suggesting that the receptor involved belongs to the 5‐HT2C subtype.

Unraveling the complexity of amyotrophic lateral sclerosis: recent advances from the transgenic mutant SOD1 mice.

Amyotrophic Lateral Sclerosis (ALS), which accounts for the majority of motor neuron disorders, is a progressive and fatal neurodegenerative disease leading to complete paralysis of skeletal muscles and premature death usually from respiratory failure. About 10% of all ALS cases are inherited, with the responsible gene having been identified in approximately 25% of these individuals. Mutations in the copper-zinc superoxide dismutase (SOD1) gene were the first to be recognized nearly twenty years ago, and since then different animal models, in particular transgenic rodents, have been developed. They replicate many of the clinical, neuropathological and molecular features of ALS patients and have contributed significantly to our understanding of the pathogenic mechanisms of this disease. Although results obtained so far with mutant SOD1 mice have not translated into effective therapies in ALS patients, these models still represent the only experimentally accessible system to study multiple aspects of disease pathogenesis and to provide proof-of-principle for the development of new therapeutic strategies. This review will examine the most recent discoveries obtained from these animal models in an attempt to elucidate the complex mechanisms of the disease. In particular it will focus on the contribution of multiple cell types in governing the disease development and progression.